Project description:Acute hepatopancreatic necrosis disease (AHPND) is a newly emerging shrimp disease that has severely damaged the global shrimp industry. AHPND is caused by toxic strains of Vibrio parahaemolyticus that have acquired a "selfish plasmid" encoding the deadly binary toxins PirAvp/PirBvp To better understand the repertoire of virulence factors in AHPND-causing V. parahaemolyticus, we conducted a comparative analysis using the genome sequences of the clinical strain RIMD2210633 and of environmental non-AHPND and toxic AHPND isolates of V. parahaemolyticus Interestingly, we found that all of the AHPND strains, but none of the non-AHPND strains, harbor the antibacterial type VI secretion system 1 (T6SS1), which we previously identified and characterized in the clinical isolate RIMD2210633. This finding suggests that the acquisition of this T6SS might confer to AHPND-causing V. parahaemolyticus a fitness advantage over competing bacteria and facilitate shrimp infection. Additionally, we found highly dynamic effector loci in the T6SS1 of AHPND-causing strains, leading to diverse effector repertoires. Our discovery provides novel insights into AHPND-causing pathogens and reveals a potential target for disease control.IMPORTANCE Acute hepatopancreatic necrosis disease (AHPND) is a serious disease that has caused severe damage and significant financial losses to the global shrimp industry. To better understand and prevent this shrimp disease, it is essential to thoroughly characterize its causative agent, Vibrio parahaemolyticus Although the plasmid-encoded binary toxins PirAvp/PirBvp have been shown to be the primary cause of AHPND, it remains unknown whether other virulent factors are commonly present in V. parahaemolyticus and might play important roles during shrimp infection. Here, we analyzed the genome sequences of clinical, non-AHPND, and AHPND strains to characterize their repertoires of key virulence determinants. Our studies reveal that an antibacterial type VI secretion system is associated with the AHPND strains and differentiates them from non-AHPND strains, similar to what was seen with the PirA/PirB toxins. We propose that T6SS1 provides a selective advantage during shrimp infections.
Project description:Acute hepatopancreatic necrosis disease (AHPND) is a severe shrimp disease originally shown to be caused by virulent strains of Vibrio parahaemolyticus (VPAHPND). Rare cases of AHPND caused by Vibrio species other than V. parahaemolyticus were reported. We compared an AHPND-causing V. campbellii (VCAHPND) and a VPAHPND isolate from the same AHPND-affected pond. Both strains are positive for the virulence genes pirABvp . Immersion challenge test with Litopenaeus vannamei indicated the two strains possessed similar pathogenicity. Complete genome comparison showed that the pirABvp -bearing plasmids in the two strains were highly homologous, and they both shared high homologies with plasmid pVA1, the reported pirABvp -bearing plasmid. Conjugation and DNA-uptake genes were found on the pVA1-type plasmids and the host chromosomes, respectively, which may facilitate the dissemination of pirABvp . Novel variations likely driven by ISVal1 in the genetic contexts of the pirABvp genes were found in the two strains. Moreover, the VCAHPND isolate additionally contains multiple antibiotic resistance genes, which may bring difficulties to control its future outbreak. The dissemination of the pirABvp in non-parahaemolyticus Vibrio also rises the concern of missing detection in industrial settings since the isolation method currently used mainly targeting V. parahaemolyticus. This study provides timely information for better understanding of the causes of AHPND and molecular epidemiology of pirABvp and also appeals for precautions to encounter the dissemination of the hazardous genes.
Project description:A strain of Vibrio (KC13.17.5) causing acute hepatopancreatic necrosis disease (AHPND) in shrimp in northern Vietnam was isolated. Normally, AHPND is caused by Vibrio parahaemolyticus, but the genomic sequence of the strain indicated that it belonged to Vibrio harveyi. The sequence data included plasmid-like sequences and putative virulence genes.
Project description:The acute hepatopancreatic necrosis disease (AHPND) of Penaeus vannamei shrimp is caused by Vibrio parahaemolyticus carrying toxin genes, pirA and pirB We report the complete genome sequence of the novel V. parahaemolyticus strain R14, which did not display AHPND symptoms in P. vannamei despite containing the binary toxin genes.
Project description:Acute hepatopancreatic necrosis disease (AHPND) has caused sharp declines in aquaculture industries of whiteleg shrimp Penaeus vannamei in Asia and the Americas since 2010. Vibrio parahaemolyticus, V. campbellii, V. owensii, and V. punensis have been proved to cause AHPND. However, the mechanisms underlying the burgeoning number of Vibrio species that cause AHPND is not known. All of AHPND-causing Vibrio bacteria (V AHPND) harbor a highly homologous plasmid (designated as pVA1-type) carrying pirAB vp toxin genes. In this study, we demonstrate conclusively that the pVA1-type plasmid can be transferred from V AHPND to non-pathogenic bacteria. We constructed a pVPGX1-Cm r plasmid (a pVA1-type plasmid) by adding a chloramphenicol resistance gene as a marker in a donor AHPND-causing V. parahaemolyticus 20130629002S01 (Vp2S01). Horizontal transfer of this plasmid was successfully performed from the AHPND-Vp2S01 to a non-pathogenic strain of V. campbellii at the transfer efficiency of 2.6×10-8 transconjugant/recipient, and DNase I treatment did not eliminate the transfer. The recipient V. campbellii acquired the pVA1-type plasmid and was shown to produce pirAB vp RNA and proteins. Challenge studies using the transconjugant caused 100% mortality in exposed groups of P. vannamei. The challenged shrimp, infected with the transconjugant bacteria, showed typical gross signs and histological lesions of AHPND. These results demonstrated the conjugative transfer of an AHPND pVA1-type plasmid. It provides timely information for explaining the increased species of AHPND-causing Vibrio bacteria and will be useful in the development of management strategies leading to the prevention and control of AHPND.
Project description:The halophilic aquatic bacterium, Vibrio parahaemolyticus, is an important aquatic pathogen, also capable of causing acute hepatopancreatic necrosis disease (AHPND) in shrimp resulting in significant economic losses. Therefore, there is an urgent need to develop anti-infective strategies to control AHPND. The gnotobiotic Artemia model is used to establish whether a phenolic compound phloroglucinol is effective against the AHPND strain V. parahaemolyticus MO904. We found that pretreatment with phloroglucinol, at an optimum concentration (30?µM), protects axenic brine shrimp larvae against V. parahaemolyticus infection and induced heat shock protein 70 (Hsp70) production (twofolds or more) as compared with the control. We further demonstrated that the Vibrio-protective effect of phloroglucinol was caused by its prooxidant effect and is linked to the induction of Hsp70. In addition, RNA interference confirms that phloroglucinol-induced Hsp70 mediates the survival of brine shrimp larvae against V. parahaemolyticus infection. The study was validated in xenic Artemia model and in a Macrobrachium rosenbergii system. Pretreatment of xenic brine shrimp larvae (30?µM) and Macrobrachium larvae (5?µM) with phloroglucinol increases the survival of xenic brine shrimp and Macrobrachium larvae against subsequent V. parahaemolyticus challenge. Taken together, our study provides substantial evidence that the prooxidant activity of phloroglucinol induces Hsp70 production protecting brine shrimp, A. franciscana, and freshwater shrimp, M. rosenbergii, against the AHPND V. parahaemolyticus strain MO904. Probably, phloroglucinol treatment might become part of a holistic strategy to control AHPND in shrimp.
Project description:Acute hepatopancreatic necrosis disease (AHPND) is an emerging penaeid shrimp disease caused by Vibrio parahaemolyticus. Although V. parahaemolyticus has been isolated and sequenced from several Asia countries, the epidemiological links among the AHPND outbreaks in different locations remain unclear. In this study, we sequenced the genomes of nine strains isolated in China between 2014 and 2016 from four sampling sites in three provinces. Analysis of single nucleotide polymorphisms (SNPs) among the nine isolates yielded an average of 35,519 SNPs per isolate, ranging from 35,001 SNPs to 35,889 SNPs relative to the reference genome FDA_R31. To capture the genetic diversity of V. parahaemolyticus in Asia and Mexico, 93 published genomes were included in the analysis. Phylogenetic analysis divided the 102 isolates into 5 clades from I to V, with the majority belonging to Clade I and Clade II. There were at least 12 independent AHPND related clones. The results indicated that the clones recovered from AHPND affected shrimps in Asia were genomically distinct in various locations and there are no epidemiological links between Asian and Mexico outbreaks. Core genome analysis of pVA-1-like plasmid sequences from V. parahaemolyticus revealed that the AHPND-associated plasmids were also genetically diverse. Homology analysis of the publicly available microbial genomes showed that the conjugative transfer gene clusters of the plasmids in AHPND-causing strains were found in 27 V. parahaemolyticus strains and several other Vibrio sp. from 10 countries including five strains isolated prior to the first identification of AHPND outbreak, indicating that the backbone of AHPND- associated plasmid was widely distributed around the globe. In conclusion, at least 11 origins of AHPND outbreaks were identified; as AHPND-causing plasmid is widely distributed globally, prevention strategies for AHPND need to focus on microbial management in the aquaculture system and establishing ecological friendly aquaculture practices instead of detection of plasmid alone. However, more strains from other Asia countries as well as Mexico need to be included for whole genome sequencing (WGS) for reconstruction of the global transmission and the spread patterns of AHPND.
Project description:Acute hepatopancreatic necrosis disease (AHPND) is an emerging shrimp (Penaeus monodon) disease caused by Vibrio parahaemolyticus (VP) since 2013 in Bangladesh. The aim of this work was to evaluate a PCR and RT-PCR techniques as rapid methods for detecting V. parahaemolyticus AHPND-positive P. monodon using genetic markers. Healthy and diseased shrimp (P. monodon) samples were collected from three monitoring stations. The samples were enriched in TCBS plates and DNA extraction from the cultured bacteria. DNA quantifications, PCR amplification, RT-PCR, and gene sequencing were done for the detection of V. parahaemolyticus AHPND-positive P. monodon. The sequence of PCR amplicons showed 100% identity and significant alignment with V. parahaemolyticus. The primers used provided high specificity for V. parahaemolyticus in PCR detection compared with another Vibrio species. In the PCR, amplification resulted positive amplicons, whereas, non-AHPND isolates showed no amplicons. Neighbor-joining methods indicated that all genes evolved from a common ancestor and clades have different traits with very low genetic distance and low variability. The pairwise alignment scores of atpA, tox, blaCARB, 16S rRNA, and pirA genes were 100.0, 98.90, 98.89, 95.53, and 41.42, respectively. The RT-qPCR exposed variable expression levels for all genes in the AHPND-positive strain. Homology analysis and distance matrix exhibited all genes to have the lowest similarity and most divergence, offering the highest specificity. In this study, the expression and variability of target genes confirmed the presence of V. parahaemolyticus in all sampling sites. The results suggested that PCR amplification, RT-qPCR, and gene sequencing can be used for the rapid detection of V. parahaemolyticus in AHPND-positive P. monodon that may lead to subsequent prevention and treatment research in the future for managing this disease.
Project description:Some strains of Vibrio parahaemolyticus cause acute hepatopancreatic necrosis disease (AHPND) in shrimp. We sequenced 3 AHPND and 3 non-AHPND strains and found that all of them lacked the pathogenicity island relevant to human infection. A unique sequence encoding a type IV pilus/type IV secretion system was found in 3 AHPND strains.
Project description:The biofloc system is a relatively new aquaculture technology that offers practical solution to maintain culture water quality by recycling nutrients and improves the health status and resistance of shrimps against microbial infection, yet the mode of action involved remains unclear. This study aimed to unravel the underlying mechanism behind the protective effect of a biofloc system using Litopenaeus vannamei and acute hepatopancreatic necrosis disease (AHPND)-causing Vibrio parahaemolyticus M0904 strain as a host-pathogen model. The results showed that a biofloc system maintained at a C/N ratio of 15, improves the water quality and contributes to the nutrition of cultured animals as bioflocs might serve as an additional protein source. Furthermore, the study demonstrated that the biofloc system enhances the survival of L. vannamei upon challenge with a V. parahaemolyticus AHPND strain. Remarkably, the results highlight that in the biofloc system, AHPND-causing V. parahaemolyticus possibly switch from free-living virulent planktonic phenotype to a non-virulent biofilm phenotype, as demonstrated by a decreased transcription of flagella-related motility genes (flaA, CheR, and fliS), Pir toxin (PirB VP ), and AHPND plasmid genes (ORF14) and increased expression of the phenotype switching marker AlkPhoX gene in both in vitro and in vivo conditions. Taken together, results suggest that biofloc steer phenotype switching, contributing to the decreased virulence of V. parahaemolyticus AHPND strain toward shrimp postlarvae. This information reinforces our understanding about AHPND in a biofloc setting and opens the possibility to combat AHPND not only by trying to eliminate the AHPND-causing V. parahaemolyticus from the system but rather to steer the phenotypic switch.